The overexpression of long intergenic ncRNA00162 induced by RelA/p65 promotes growth of pancreatic ductal adenocarcinoma.

Abstract Objectives Recent observations have emphasized the role of long non‐coding RNA (lncRNA) in cancer progression; however, a genetic profile of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) remains an ongoing study. Materials and Methods In this research, RNA sequencing showed that LINC00162 is dramatically increased in patient‐derived tumour cell lines (PATC) compared with the human pancreatic nestin‐positive epithelial (HPNE) cells. Results These data were validated in several PDAC cell lines, and significant upregulation of LINC00162 was found in all of them. Knock‐down of LINC00162 significantly inhibited the proliferation, colony formation and migration of PATC cells in vitro and suppressed the growth of PATC xenografts in vivo. Overexpression of LINC00162 in PDAC cell lines (AsPc‐1) showed consistent results, with significantly increased proliferation, colony formation and migration of AsPc‐1 cells, as well as enhanced tumour growth of the AsPc‐1 xenografts in vivo. Furthermore, the result of Chromatin immunoprecipitation assay revealed that RelA/p65 directly bound to LINC00162, and the expression of LINC00162 in PDAC decreased after RelA/p65 knock‐down, the proliferation ability of AsPc‐1 also significantly inhibited after knocking down LINC00162 and RelA/p65 simultaneously, indicating that RelA/p65 directly involve in the transcriptional regulation of LINC00162. Conclusions In sum, our results provide first evidence for the role of LINC00162 in promoting PDAC progression and the potential underlying mechanism of LINC00162 overexpression.

biomarkers will help us better understand this disease and develop effective targeted therapies.
Advances in large scale genome analyses have identified several most commonly detected mutations and alterations as the signatures of PDAC, 2 such as KRAS mutation-an early event in PDAC development that has been detected in 80%-95% of PDAC; inactivation of Ink4a and p53 tumour-suppressor genes, identified by Hruban in approximately 50%-75% of PDAC cases; and the loss of CYLD, identified by Vogelstein's lab in a comprehensive genetic analysis of 24 PDACs. 3 Among all these findings, most of the researchers focus on the protein-coding genes. However, according to the genome data, sequences of protein-coding genes comprise <3% of the human genome, while the majority of the remaining genomic sequences are transcribed into non-coding RNAs, indicating that the human transcriptome consists largely of non-coding RNAs. 4 Recently, long non-coding RNAs (lncRNAs), which exceed 200 nucleotides in length, have attracted more attention in cancer research and increasing evidence has suggested that lncRNAs constitute an important component of tumour biology. For instance, by reprogramming the chromatin state, the overexpression of the lncRNA HOTAIR was found to promote the invasiveness and metastasis of breast cancer cells, and its expression level in primary tumours was determined to be a potent predictor of final metastasis and death in breast cancer. 5 The lncRNA PCAT19 also interacts with HNRNPAB to activate a subset of cell-cycle genes associated with prostate cancer progression, thus promoting prostate cancer growth and metastasis. 6 Guo et al 7 found that lncRNA SNHG6 is not only an oncogene in hepatocarcinogenesis but also a potential prognostic indicator for hepatocellular carcinoma (HCC), and, by inhibiting S-adenosylmethionine production in HCC, dysregulation of SNHG6 can lead to aberrant genome-wide hypomethylation and further disturb the treatment of HCC. Therefore, lncRNAs are pervasively transcribed and contribute to the hallmarks of various cancers. 4 However, to the authors' knowledge, few studies consider lncRNAs expression profiles in PDAC, and the potential clinicopathological significance of lncRNAs in PDAC remains unclear.
In the present study, a genetic profile of lncRNA in PDAC was carried out. The researchers screened gene expression profiles in four cells established from patient-derived xenografts of PDAC, as well as hTERT-immortalized human pancreatic epithelial nestin-expressing cells (HPNE) by RNA sequencing. Using this data, the expression of selected lncRNAs was further validated and integrated functional studies were conducted. This study aimed at providing an in-depth understanding of lncRNA in carcinogenesis and identifying clinically relevant targets for PDAC.

| Cell lines and culture
The human pancreatic cancer cell lines (AsPc-1, BxPc-3, Panc-1, Pan-28 and Miapaca 2) were purchased from the American Type Culture Center Core Facility. The comparison of the lncRNA expression profiles among these groups was performed using the tophat2 and cuffdiff standard procedure. The lncRNAs with at least 2.0-fold changes and P < .05 between the study and control groups were considered significantly different expressions.

| Fluorescence in situ hybridization (FISH)
LncRNA expression was evaluated by FISH using Stellaris ® RNA FISH Probes and Methods (LGC Biosearch Technologies). Custom Stellaris ® FISH Probes were designed against LINC00162 (NC_000021.9) by utilizing the Stellaris ® RNA FISH Probe Designer (Biosearch Technologies, Inc) available online at www.biose archt ech.com/stell F I G U R E 1 Expression of LINC00162 is specially increased in PATC and PDAC cells. A, The heat map of an RNA sequencing study showed significantly upregulated lncRNA in PATC cells when compared with HPNE, with LINC00162 being the most upregulated; (B) The differential expression of LINC00162 was further validated in a series of PATC and PDAC cells, and an upregulation of LINC00162 was found in all of them, compared with HPNE; (C) FISH results showed that LINC00162 is mainly located in cytoplasm; (D) Higher signals of LINC00162 were observed in PATC cells, but not in HPNE cells, by RNA FISH arisd esigner. The PDAC cells were hybridized with the LINC00162 Stellaris ® RNA FISH Probe set labelled with Quasar ® 570 Dye (Biosearch Technologies, Inc), following the manufacturer's instructions available online at www.biose archt ech.com/stell arisp rotocols.
Briefly, PDAC cells were grown on 18 mm round coverglass in a 12well cell culture plate. They were fixed with fixation buffer for ten minutes and permeabilized in 70% ethanol for 1 hour before being hybridized overnight with a 125 nmol/L LINC00162 specific probe set at 37°C. 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used for nuclear staining. The fluorescence signals of LINC00162 and DAPI were observed using Zeiss LSM 710 (Carl Zeiss).

| Lentiviral transfection
Human lentiviral shRNA against human LINC00162 was designed.
The sequence for LINC00162 was 5′-GCTCTATGACCTCATCCAT-3′ AsPc-1 was used to establish LINC00162 high-level expression cell lines, where it was transfected by the lentivirus expressing LINC00162 and GFP with puromycin resistance. The following lentivirus production, infection procedures, and drug selections were performed as described above for the lentivirus. The number of clones (≥50 cells) was observed and counted manually under light microscopy. Each experiment was performed in triplicate.

| Cell migration assay
Cell migration assay was performed using a Transwell chamber with a polycarbonate membrane containing 8.0-μm pores (BD Biosciences).
Cells were resuspended in serum-free medium and plated in the upper chamber at a concentration of 5 × 10 4 cells/well. The upper chamber was then inserted into a well of a 24-well plate containing 600mL serum-containing medium (20%). After incubation for 24 hours, the nonmigrating cells in the upper chamber were wiped with cotton swabs and removed, and those that had migrated through the pores were fixed with 4% paraformaldehyde for 15 minutes and stained with 1% crystal violet for 15 minutes. The migrated cells in each chamber were observed at 100× magnification; three random fields were chosen to count and determine the mean number for each group.

| Animal model
The orthotopic pancreatic tumour-bearing mouse model was es- Standard procedures were carried out according to the manual previously described, 14  Center.

| Chromatin immunoprecipitation (ChIP) assay
AsPc-1 cells were fixed with 1% formaldehyde for 10 minutes for the cross-link between protein and DNA, followed by quenching with

| Assessment of LINC00162 Expression in -RelA/p65 knock-down cells and rescues experiment
RelA/p65 knock-down cells AsPc-1 were established as mentioned above, and the expression of LINC00162 in these cells was detected using qRT-PCR and FISH. A rescue experiment was also performed by knocking down LINC00162 and RelA/p65 simultaneously to verify the regulation between RelA/p65 and LINC00162.

| Statistical analysis
For error bars in all experiments, the standard deviation (sd) was calculated from three independent experiments, and the data are expressed as mean ± sd. For comparison of the significant differences among more than two groups, one-way ANOVA and Newman Keul's multiple comparison tests were used, and other differences were evaluated by the Student unpaired t-test. All statistical analyses were performed using a commercially available statistical software package (SPSS for Windows, 16.0), and differences reached statistical significance with P < .05.  Figure 1B). To detect the location of LINC00162, RNA FISH was used. The results showed that LINC00162 was mainly located in cytoplasm ( Figure 1C), and higher signal was observed in PATC cells but not in HPNE cells ( Figure 1D).

| LINC00162 affects proliferation, colony formation and migration of PATC cells
To study the cellular functions of LINC00162, two PATC cell lines and one PDAC cell line were selected to carry out lossof-function and gain-of-function experiments, respectively.

| LINC00162 affects growth of PDAC xenografts in vivo
The role of LINC00162 in PDAC progression in vivo was in- were injected, the mass of the LINC00162 overexpression tumour was significantly higher compared to the control tumours ( Figure 4C).

| RelA/p65 directly involves in the transcriptional regulation of LINC00162
To Then, further exploration by knocking down RelA/p65 ( Figure 6A) to evaluate the alteration of LINC00162 was also carried out. As shown in Figure 6B Figure 6D). Collectively, these results indicate that RelA/p65 directly involves in the transcriptional regulation of LINC00162.

| D ISCUSS I ON
So far, more than 11 000 lncRNA genes and 18 000 transcripts have been identified 15 23 is also found constitutively activated in nearly 70% of PDAC tumours and most PDAC cell lines. 2 Since our lab has previously showed that the NF-κB pathway is an important downstream pathway of KRAS mutation, and inhibition of NF-κB activity results in the inhibition of PDAC cell tumorigenesis, 24,25 this study has sought to confirm if there is any relationship between LINC00162 expression and NF-κB. As shown in Figure 5, the result of ChIP assay revealed that RelA/p65 directly bound to LINC00162, and further knock-down of RelA/p65 led to significantly decreased expression of LINC00162, indicating that the expression of LINC00162 was induced by RelA/p65; the proliferation ability of AsPc-1 also significantly inhibited after knocking down LINC00162 and RelA/p65 simultaneously. Collectively, all these finding indicated that RelA/p65 directly involves in the transcriptional regulation of LINC00162.
In sum, this study has identified LINC00162 as a novel lncRNA, differentially overexpressed in PATC and PDAC cells. In addition, it has found that LINC00162 regulates the proliferation, colony formation and migration of PATC and PDAC cells in vitro, as well as their growth in vivo. These results provide mechanistic evidence that LINC00162 is induced by RelA/p65, and all these findings indicate that LINC00162 is involved in promoting the growth of PDAC and may act as a biomarker and putative therapeutic target in PDAC.

ACK N OWLED G EM ENTS
The authors thank Dr Jun Yao of the department of Molecular and Cellular Oncology, MD Anderson Cancer Center, for the data analysis of RNA sequences.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
PC and XQ conceived and designed the study. YL and MW per-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.